The purpose of this project is to define the structure of the antigenic site(s) associated with variance in antigenic determinants on the rabies virus glycoprotein. The importance of this work can be clearly seen when one realizes that vaccine strains and field strains of rabies virus now show differences in antigenic composition of the glycoprotein. The molecular basis for antigenic variation in rabies virus glycoprotein will be studied at the level of the glycoprotein gene. Small restriction DNA fragments will be prepared from our cloned, ERA strain, glycoprotein gene cDNA and used to prime the synthesis of cDNA transcripts with reverse transcriptase and rabies virus genome RNA (vRNA) as template. Using predominantly the "dideoxy" DNA sequencing method, the nucleotide sequence of various virus glycoprotein gene will be determined from overlapping partial sequences of the elongated cDNA primers. A physical map of the major antigenic determinants on the rabies virus glycoprotein will be constructed from the analysis of gene mutations in laboratory-selected antigenic variants of the ERA strain of rabies virus. This will be aided by an epitope map, which operationally defines antigenic areas on the glycoprotein. By comparing partial cDNA sequences from specific regions of the glycoprotein gene of several unique antigenic variants, it will be possible to identify and map nucleotide substitutions associated with high antigenic variation in the ERA strain. To study the structural requirements for cross-reactivity between vaccine strains, the glycoprotein gene of other laboratory strains of rabies virus and rabies-related viruses will be examined. Since the long range goal of this project is to assess the extent of antigenic variation in field strains of rabies virus, it is important to study first the relative variability of laboratory strains, to more accurately define the significance of the observed antigenic variance in field strains.